I plan to use zebrafish to study the formation of the floor plate, an important organizing center in the ventral neural tube. Floor plate is induced by Sonic hedgehog (Shh) secreted by the notochord. However, recent work indicates that the notochord is not always required for floor plate formation. For example, in zebrafish no tail (ntl) mutants, notochord fails to differentiate, but the width of the floor plate is actually expanded. Expression of hedgehog (hh) related genes is also altered. In wild type embryos, shh and tiggy-winkle hedgehog (twhh) are coexpressed in cells of the embryonic shield (the equivalent of the Xenopus organizer). During gastrulation, shh is coexpressed with Ntl protein in a layer that gives rise to notochord; whereas twhh is expressed in an adjacent layer that is presumed to give rise to floor plate. In ntl mutants, shh and twhh expression do not segregate to separate layers during gastrulation. One hypothesis is that Ntl regulates a choice between notochord and floor plate fates, possibly by influencing hh gene expression during gastrulation. I will use molecular, cellular and genetic techniques available in the zebrafish to test the following specific hypotheses: (1) the twhh expressing cells of the gastrula solely give rise to floor plate (2) the twhh and shh coexpressing cells of the embryonic shield have the potential to give rise to floor plate (2) the twhh and shh coexpressing cells of the embryonic shield have the potential to give rise to either notochord or floor plate depending upon their position (3) the Ntl transcription factor promotes notochord differentiation and represses floor plate development and (4) twhh is essential for floor plate specification.